3-aminopyrazolo heterocyclic derivatives, and use for colorimetric determinations

ABSTRACT

The present invention is concerned with the use of certain 3-aminopyrazolo-heterocyclic compounds for the colorimetric determination of hydrogen peroxide, hydrogen peroxide-forming systems, peroxidase, peroxidate-active substances and electron-rich aromatic compounds. The present invention is also concerned with corresponding processes of determination and with agents appropriate therefor. Furthermore, the present invention is concerned with new 3-aminopyrazolo-heterocyclic compounds.

The present invention is concerned with the use of3-aminopyrazolo-heterocyclic compounds for the colorimetricdetermination of hydrogen peroxide, hydrogen peroxide-forming systems,peroxidase, peroxidate-active substances and of electron-rich aromaticcompounds, as well as with new 3-aminopyrazolo-heterocyclic compoundsand a process for the preparation thereof.

Furthermore, the present invention is concerned with a process for thecolorimetric determination of hydrogen peroxide, hydrogenperoxide-forming systems, peroxidase and peroxidate-active substances bymeans of oxidative coupling of an electron-rich aromatic compound with aheterocyclic compound.

In addition, the present invention is also concerned with an agent forthe colorimetric determination of hydrogen peroxide, hydrogenperoxide-forming systems, peroxidase and peroxidate-active substances byoxidative coupling, containing an electron-rich aromatic compound and aheterocyclic compound.

The present invention is also concerned with the use of a3-aminopyrazolo derivative for the production of an agent for thecolorimetric determination of hydrogen peroxide, hydrogenperoxide-forming systems, peroxidase and peroxidate-active substances byoxidative coupling.

The present invention is also concerned with a process for thecolorimetric determination of an electron-rich aromatic compound by theoxidative coupling thereof with a heterocyclic compound in the presenceof an oxidation agent.

In addition, the present invention is also concerned with an agent forthe colorimetric determination of an electron-rich aromatic compound byoxidative coupling, containing a heterocyclic compound and an oxidationagent.

Finally, the present invention is concerned with the use of a3-aminopyrazolo derivative for the production of an agent for thecolorimetric determination of an electron-rich aromatic compound byoxidative coupling.

Not only for analytical chemistry but also for medical diagnosis, thedetection of hydrogen peroxide by means of chromogenic substances, withthe catalysis of peroxidase or of peroxidate-active substances, forexample haemoglobin, is of very great importance. This applies inparticular to numerous detection processes in which hydrogen peroxide isformed as an intermediate and subsequently, in the presence ofappropriate chromogenic compounds, preponderantly in the presence of aperoxidase (POD), as a catalyst, is converted into a compound which canbe detected optically and is in a quantitative relationship to thehydrogen peroxide formed. Furthermore, peroxidase is frequently used asan enzyme label in immune tests and is detected by the addition ofhydrogen peroxide and the above-mentioned chromogenic substances.

As examples, the following compounds are mentioned which, with theoxidases indicated in brackets, represent hydrogen peroxide-formingsystems: glucose (glucose oxidase), galactose (galactose oxidase),L-amino acids (L-amino acid oxidase), cholesterol (cholesterol oxidase),uric acid (uricase), sarcosine (sarcosine oxidase) and glycerol(glycerol oxidase).

Numerous chromogens and indicator systems have already been describedand used for the detection of hydrogen peroxide/peroxidase. One of thebest known ones is that of Trinder (see Ann. Clin. Biochem., 6,24-27/1969), an indicator system in which phenol is oxidatively coupledwith 4-aminoantipyrine in the presence of peroxidase with the action ofhydrogen peroxide to give a coloured material. As coupling component,instead of phenol there can also be used phenol derivatives, anilinederivatives, naphthol, naphthol derivatives, naphthylamine,naphthylamine derivatives and other compounds which react in a similarmanner. 4-Aminoantipyrine as coupling component can be substituted, forexample, by aminoantipyrine derivatives, vanilindiaminesulphonic acid,methylbenzthiazolinone hydrazone (MBTH), sulphonatedmethylbenzthiazolinone hydrazone (SMBTH) and similar systems describedby S. Hunig (cf. "The Chemistry of Synthetic Dyes", K. Venkataraman ed.,Vol. IV, pp. 189-193, publ. Academic Press, New York, San Francisco,London, 1971).

In published European Patent Specification No. A-0,033,539, for theachievemnt of a better colour stability of the coloured materialresulting in the case of the oxidative coupling of 4-aminoantipyrinewith phenols, there is suggested the replacement of 4-aminoantipyrine by4-aminoantipyrine derivatives which bear a doubly substituted phenylradical. The coloured materials which can thus be obtained displayλ_(max) values in the range of about 500 to 550 nm.

In all, a great disadvantage of the previously described redox indicatorsystems based upon 4-aminoantipyrine orN-methylbenzthiazolinone-hydrazone is the fact that, by means ofoxidative coupling with electron-rich aromatic compounds, for examplephenols and anilines, coloured materials result, the λ_(max) values ofwhich lie in the region of about 500±50 nm. Oxidative coupling reactionsare frequently employed in the case of the analysis of body fluids, forexample blood, plasma, serum, urine, saliva and the like. However,especially in the case of haemolysed plasma and sera, i.e.haemoglobin-containing plasma and sera, problems arise in the case ofthe use of redox indicator systems of the prior art which enter intooxidative coupling reactions which are conditioned by the relativelyhigh inherent absorption of such sample materials in the region of about500 nm. Impairments of the measurement are then the more serious thesmaller the concentration of the serum component to be determined.

Furthermore, because of the blank value instability caused byautoxidation or of the instability of the colour produced at the pHvalues of about 6 to 8 needed for the enzymatic reaction, many of theknown redox indicators are not suitable for the development of ahydrogen peroxide determination.

This applies particularly to2-hydroxy-3-amino-5,7-dimethylpyrazolopyrimidine described, inter alia,by W. Ried and E.-V. Kocher in Liebigs Ann. Chem., 647, 116-144/1961 andin Liebigs Ann. Chem., 647, 144-154/1961 which is so unstable towardoxygen that it was not possible to obtain it as a pure compound.

In spite of the large number of known redox indicators for the detectionof hydrogen peroxide, peroxidase and peroxidate-active substance, thesearch is still being made for those compounds which can be used in asmany test systems as possible, especially for the determination ofcomponents of body fluids, which display a high sensitivity over a widepH range and which are not disturbed by serum components.

Therefore, in the scope of the objects of the present invention,appropriate compounds are to be made available which can be used for thedetermination of hydrogen peroxide, hydrogen peroxide-forming systems,peroxidase, peroxidate-active substances and of electron-rich aromaticcompounds, which, by oxidative coupling with phenols, anilines and othercouplers, give coloured materials, the absorption of which issufficiently in the long-wave region in order not to be disturbed by theinherent absorption of blood plasma or serum, which satisfy therequirements with regard to blank value stability and colour stabilityat the measurement pH and in the case of which the colour yield in thecase of the oxidative coupling is not disturbed by blood components.

Thus, the present invention is concerned with the use of pyrazoloderivatives of the general formula: ##STR1## wherein --X--Y-- signifies--NR¹ --CO-- or --N═CR² -- in which R¹ is an alkyl radical and R² isalkyl, alkenyl, alkoxy, alkylthio, aryl or aralkyl, optionallysubstituted in each case by hydroxyl, dialkylphosphinyl, carboxyl, SO₃H, PO₃ H₂, a salt of one of these acid residues and/or alkoxycarbonyl;amino, which is optionally substituted by one or two alkyl radicalswhich, in turn, are optionally substituted by one or more hydroxyl,carboxyl and/or alkoxycarbonyl radicals, when amino is substituted bytwo alkyl radicals these radicals can also be joined to give a ringwhich, apart from the nitrogen atom of the amino group, can optionallybe interrupted also by oxygen, sulphur or a further nitrogen atom oramino is optionally substituted by one or two acyl radicals, alkoxy-and/or aralkoxycarbonyl radicals, H₂ N--CO--, alkyl, aralkyl- and/orarylcarbamoyl radicals; or is hydrogen, carboxyl, alkoxycarbonyl,carboxamido or halogen and Z signifies --NR³ --N═N--, wherein R³ is analkyl or aralkyl radical or Z is an unsaturated chain containing 3 to 5members of nitrogen atoms or carbon atoms and optionally one or morenitrogen or sulphur atoms, whereby carbon atoms are optionallysubstituted by alkyl, alkoxy, alkylthio, hydroxyl, aralkyl, aryl,carboxyl, carboxamido, alkoxycarbonyl, cyano, amino, which is optionallysubstituted by one or two alkyl radicals which, in turn, are optionallysubstituted by one or more hydroxyl, carboxyl and/or alkoxycarbonylradicals, or halogen and whereby nitrogen atoms which are not connectedvia a double bond are substituted by alkyl or aralkyl and twoneighbouring chain substituents can optionally form an alkylene radicalwhich, in turn, is optionally substituted or anellated with aryl, aswell as optionally the corresponding tautomeric forms and the saltsthereof for the colorimetric determination of hydrogen peroxide,hydrogen peroxide-forming systems, peroxidase or peroxidate-activesubstances and electron-rich aromatic compounds. "Alkyl", also inalkyltio, dialkylphosphinyl, alkylcarbamoyl and aralkyl radicals, herebymeans a straight-chained or branched alkyl radicals containing up to 6and preferably up to 4 carbon atoms, the methyl, ethyl, propyl, isobutyland tert.-butyl radicals being preferred.

When an amino group is substituted by two alkyl radicals, these radicalscan also be joined to form a ring in such a manner that, in all, itrepresents a ring interrupted by a nitrogen atom. Hereby preferred arethose amino groups which, in all, represent a five- or six-membered ringand which, in turn, is optionally interrupted by oxygen, sulphur ornitrogen, the morpholino radical being especially preferred.

"Alkoxy", also in alkoxycarbonyl and aralkoxycarbonyl radicals, standsfor a straight-chained or branched alkoxy radical containing up to 6 andpreferably up to 4 carbon atoms, the methoxy, ethoxy, propoxy, isobutoxyand tert.-butoxy radicals being preferred.

"Aryl", also in arylcarbamoyl radicals, means a carbon aromatic orheteroaromatic radical, preferably one with 6 to 10 ring atoms andespecially a phenyl or naphthyl radical which can additionally also besubstituted by alkyl, alkoxy and/or halogen, the phenyl radical beingespecially preferred.

An "aralkyl radical", also in an aralkylcarbamoyl radical, means aradical in which an alkyl radical defined as hereinbefore is substitutedby an aryl radical as hereinbefore defined, the benzyl radical beingpreferred.

An "aralkoxy" radical, for example an aralkoxycarbonyl radical,designates a radical in which an alkoxy radical as defined hereinbeforeis substituted by an aryl radical as hereinbefore defined, the benzyloxyradical being preferred.

"Halogen" stands for fluorine, chlorine, bromine or iodine, fluorine andchlorine being preferred.

An acyl radical signifies the residue of a carboxylic acid which cancontain alkyl, aralkyl or aryl radicals, the acetyl, phenylacetyl andbenzoyl radicals being preferred.

By an alkylene radical is to be understood a straight-chained orbranched, saturated or unsaturated hydrocarbon chain containing 3 to 5and preferably 3 to 4 carbon atoms with two free bonding positions.Examples therefore include --CH₂ --CH═CH--, ##STR2## --(CH₂)₄ and--CH═CH--CH═CH--, the butadiendiyl radical (--CH═CH--CH═CH--) and thetetramethylene radical (--(CH₂)₄ --) being preferred. An alkenyl radicalis a straight chained or branched hydrocarbon chain containing 2 to 5carbon atoms with at least one double bond. For example the vinylradical is preferred. As dialkyl phosphine residue is to be understood##STR3## whereby alkyl has the meanings given above. Thedimethylphosphinyl residue is preferred.

As salts of SO₃ H, PO₃ H₂ and carboxyl residues, there can be usedalkali metal and alkaline earth metal salts and also ammonium salts. Byalkali metal salts are to be understood lithium, sodium, potassium,rubidium and caesium salts,. whereby lithium sodium and potassium saltsand especially sodium and potassium salts are preferred. Alkaline earthmetals salts are those of beryllium, magnesium, calcium, strontium orbarium, the magnesium and calcium salts being preferred and the calciumsalts being especially preferred. As ammonium salts, there can be usedthe unsubstituted ammonium ion NH₄ ⁺. However, it is also possible touse those ammonium salts in which the ammonium ion is substituted by upto 4 alkyl, aryl or aralkyl radicals. For these radicals, there applythe previously given definitions, whereby as alkyl radicals the methyl,ethyl and n-propyl radicals are preferred, as aryl radical the phenylradical and as aralkyl radical the benzyl radical are especiallypreferred.

As carboxamido radical is to be understood the CONH₂ radical but alsothose radicals in which the amino group is substituted by one or twoalkyl radicals which optionally contain one or more hydroxyl, carboxyland/or alkoxycarbonyl radicals.

In the pyrazolo derivatives used according to the present invention ofgeneral formula (I), Z is preferably so positioned that at least onedouble bond of the unsaturated chain stands in conjugation with thedouble bond or with the nitrogen atom in general formula (I).

For a compound of general formula (I), tautomeric forms can also bepossible. These are also to be regarded as being covered by generalformula (I).

The 3-aminopyrazolo derivatives of general formula (I) can be used asfree amines. Preferably, however, they are used as the correspondingammonium salts. For this purpose, salts with the most varied acids arepossible. Especially preferred are those amines of general formula (I)which are present as salts with a mineral acid, for example hydrochloricacid, sulphuric acid, phosphoric acid or nitric acid. The hydrochloridesof the compounds of general formula (I) have proved to be outstandinglyuseful.

According to the present invention, preferred compounds are those of thefollowing general formulae (II) to (XIII): ##STR4## as well as possiblythe corresponding tautomeric forms and the salts thereof.

X--Y and R³ hereby have the same meanings as given herein-before. R⁴,R⁵, R⁶ and R⁷, which can be the same or different, stand for hydrogen,hydroxyl, alkyl, alkoxy, alkylthio, aralkyl, aryl, carboxyl,alkoxycarbonyl, carboxamido, cyano, amino, which is optionallysubstituted by one or two alkyl radicals which, in turn, are optionallysubstituted by one or more hydroxyl, carboxyl and/or alkoxycarbonylradicals, or halogen, whereby two neighbouring radicals optionally forman alkylene radical which, in turn, is optionally substituted oranellated with aryl. The definitions of the radicals correspond to thosegiven for the substituents in general formula (I).

Especially preferred for the use according to the present invention arecompounds of the general formulae (II), (III), (IV), (VII) and (IX),optionally the corresponding tautomeric forms and the salts thereof.Quite especially preferred are those compounds in which X--Y has themeaning --N═CR² --, in which R² can have the meaning given in generalformula (I).

The compound 3-amino-2-methylpyrazolo[1,5-a]pyridine and the saltsthereof, especially the hydrochloride, has proved to be especiallyoutstandingly useful for the purpose of the present invention.

Some compounds of general formula (I) are known from the prior art astherapeutically-active compounds. Thus, in published Federal Republic ofGermany Patent Specification No. A-22 57 547 and in J. Med. Chem., 17,645/1974, 3-amino-5,7-dimethylpyrazolo[1,5-a]pyrimidines are describedas being 3',5'-cyclo-AMP phosphodiesterase inhibitors. In publishedFederal Republic of Germany Patent Specification No. A-30 19 019 aredescribed aminopyrazolo[1,5-c]quinazoline derivatives as compounds withpain-killing or analgesic properties which inhibit the secretion ofgastric acids and reduce the intestinal activity or displayantiperistaltic actions. In published European Patent Specification No.A-0,299,209 are described 3-aminopyrazolopyridines with diuretic,hypotensive, vasodilatory, cardiotonic and the aggregation of bloodplatelets inhibiting action. Finally, from Chemical Abstratcts, 89, 627,43340r/1978, there is known a tetracyclic 3-aminopyrazolotriazinederivative.

The use of pyrazolo derivatives of general formula (I) as components inoxidative coupling reactions for the colorimetric detection of hydrogenperoxide, hydrogen peroxide-forming systems, peroxidase,peroxidate-active substances or of electron-rich aromatic compounds isnot described in the above-mentioned prior art.

For the carrying out according to the present invention of thecolorimetric determination of hydrogen peroxide, hydrogenperoxide-forming systems, peroxidase or peroxidateactive substances bymeans of oxidative coupling, an electron-rich aromatic compound must bereacted with one of the above-described pyrazole derivatives. For bettercharacterisation, in the following, the pyrazolo derivatives accordingto the present invention are referred to as coupling components and theelectron-rich aromatic compounds necessary for the oxidative couplingare referred to as couplers.

As couplers which are able to enter into an oxidative coupling with acoupling component according to the present invention, there can, inprinciple, be used any compound which can also enter into such areaction with p-phenylenediamine as coupling component. For an expert,this is easy to test. For this purpose, a series of compounds are known,for example from colour photography. Thus, there can be used thephenolic couplers and couplers with active methylene groups mentioned byT. H. James in "The theory of the photographic process", 3rd ed., pub.Macmillan, N.Y., 1966, Chapter 17 ("Principles and Chemistry of ColorPhotography"), pp. 382-396, which enter into an oxidative coupling withthe pyrazolo derivatives according to the present invention. Thecommonest are phenols, phenol derivatives, naphthol, naphtholderivatives, naphthylamine, naphthylamine derivatives and anilinederivatives.

Preferred couplers in the meaning of the present invention areanilinophosphonic acid derivatives such as are described in publishedEuropean Patent Specification No. 0,175,250, N,N-dimethylaniline,2,4,6-tribromo-3-hydroxybenzoic acid andN-ethyl-N-3-sulpho-2-hydroxypropyl-m-anisidine.

The detection readction according to the present invention, which leadsto the formation of a coloured material, can, using the example of anN,N-dimethylaniline as coupler, be illustrated as follows: ##STR5##

Expressed generally, it can be seen herefrom that, for the detection ofhydrogen peroxide or of a system producing this, a pyrazolo derivativeof general formula (I), a coupler and peroxidase or a peroxidate-activesubstance must be brought into contact with the sample to beinvestigated. By hydrogen peroxide-producing systems are to beunderstood especially the substrate/substrate oxidase pairs which areimportant in clinical diagnosis of which, by way of example, some of themore important representatives have already been mentioned hereinbeforeand in the case of which the substrate is oxidised in the presence ofatmospheric oxygen and hydrogen peroxide is produced. Thus, eithersubstrate or substrate oxidase can be detected therewith, depending uponwhich component is known and, together with the other detectionreagents, is to be contected with the sample to be investigated.

If the enzyme peroxidase or a peroxidate-active substance is to bedetermined, the sample must be reacted with coupler, pyrazolo derivativeof general formula (I) and hydrogen peroxide. Alternatively, of course,a substance can also be detected which can act as coupler like, forexample, N,N-dimethylaniline in Scheme 1 above. A pyrazolo derivative ofgeneral formula (I) and an oxidation agent, which does not necessarilyhave to be the system hydrogen peroxide/peroxidase but can also beanother oxidation agent, for example a peroxide, such as a persulphateor peracetate, or a periodate, chloramine T or especially a cyanoferriccomplex, for example potassium ferricyanide or an oxidase according topublished Federal Republic of Germany Patent Specification No. A-33 31588, must then be brought into contact with sample.

The colour formed in the case of a positive result can then be used withregard to its intensity as a measure for the amount of the substance tobe determined. It can be evaluated visually or photometrically.

With the substances necessary for carrying out the process according tothe present invention for the colorimetric determination of hydrogenperoxide, hydrogen peroxide-forming systems, peroxidases,peroxidate-active substances or electron-rich aromatic compounds, therecan be produced agents according to the present invention which can bemeasured in a cuvette. According to the present invention, agents forthe determination of hydrogen peroxide, hydrogen peroxide-formingsystems, peroxidase or peroxidate-active substances contain anelectron-rich aromatic compound and, as heterocyclic compounds which canbe oxidatively coupled therewith, a pyrazolo derivative of generalformula (I). Separately or together with these compounds, there can bepresent further materials for the detection of the parameter inquestion, for example peroxidase for the determination of hydrogenperoxide; peroxidase and enzyme substrate for the determination of theenzyme oxidising the substrate with the formation of hydrogen peroxide;peroxidase and oxidising enzyme for the determination of the enzymesubstrate oxidisable by the enzyme with the formation of hydrogenperoxide; or hydrogen peroxide for the determination of peroxidase orperoxidate-active substances. Agents for the colorimetric determinationof an electron-rich aromatic compound contain an oxidation agent and apyrazolo derivative of general formula (I). Further components of theagent according to the present invention can be buffers, as well aspossibly wetting agents, activators and other adjuvants.

The reagent components can be pressed into tablets together, separatelyor, depending on compatibility and expediency, combined with one anotheras powder or preferably dissolved in water or a buffer solution andoptionally subsequently dried or lyophilised to give an agent accordingto the present invention.

A reagent mixture obtained in this manner is, before use, dissolved inwater or some other appropriate solvent and the reagent solution thusprepared. After mixing the sample (for example substrate solution,enzyme solution, serum or plasma) with an aliquot of the reagentmixture, the resultant colour is measured in a photometer and, via themolar extinction coefficient and the added volume or reagent or sample,the concentration or substrate concentration in question thencalculated. Kinetic as well as end-point measurements are possible.

Agents according to the present invention can contain the pyrazoloderivatives useable according to the present invention, together withthe other substances necessary for the detection of the parameter inquestion, for example oxidation agents, enzymes, electron-rich aromaticcompounds and/or enzyme substrates, buffers, optionally wetting agentsand activators, as well as other adjuvants also on or in swellable orabsorbent reagent carriers, such as polymer films, papers, membranes,fleece and the like. For this purpose, depending upon compatibility andexpediency, one or more solutions can be prepared in the form of aqueousor organic or mixed solutions, depending upon how the reagents oradjuvants dissolve. Absorbent glass or synthetic material fleece areimpregnated or sprayed with these solutions. Subsequently, a drying stepis carried out. The reagent carriers thus produced can be used either asrapid diagnostic agents for the direct determination of componentmaterials of liquids (for example in body fluids, such as blood, urineor saliva, or in foodstuffs, such as fruit juices, milk or the like).The liquid is thereby applied directly on to the reagent carrier or thereagent carrier is briefly dipped into the liquid. A semi-quantitativedetermination is also possible by associating the resultant colour witha comparison colour. A quantitative evaluation can be carried out byremission photometry. By elution of the above-mentioned reagents withwater, buffer or serum from the absorbent carrier, a reagent solutioncan be prepared with which, as described above, substrates or enzymescan be determined in a cuvette with the use of a photometer. Thequantitative determination by remission-photometric evaluation can becarried out especially well when a pyrazolo derivative useable accordingto the present invention, together with the remaining necessary reagentsand adjuvants, and a film-forming synthetic material is worked up togive a reagent film, for example according to Federal Republic ofGermany Patent Specification No. C-15 98 153. The smooth surface of sucha film thereby gives much less disturbances of the remission and a morehomogeneous coloration than the absorbent papers usually employed.

Buffers in the meaning of the present invention have a pH value of 5 to10 and especially of 6.5 to 8. Phosphate, citrate, borate and GOODbuffers with alkali metal or ammonium counterions are most frequentlyused but other systems can also be employed.

Wetting agents are especially anionic and cationic ones. Non-ionicwetting agents which activate enzymes can also be employed. Sodiumlauryl sulphate, dioctyl sodium sulphosuccinate and alkylaryl polyetheralcohols are preferably used.

In the case of the determination of an enzyme or enzyme substrate as anexample of a hydrogen peroxide-forming system, as activators there canbe used those known for the enzyme reaction in question. Thecolour-forming reaction is often so quick that an additional activationdoes not appear to be necessary.

As other adjuvants, it can be helpful to use conventional thickeners,emulsifiers, optical brighteners, contrast agents and the like, such asare known in corresponding tests with other chromogens.

The coupling reaction usually takes place at ambient temperature but canalso be carried out without difficulty at a higher temperature, forexample at 37° C., if this appears to be desirable for the reactionvelocity of, for example, a preceding enzymatic reaction.

For the reactions with enzymes oxidising substrates or with substrateswhich usually occur, the following concentrations of the test solutionhave proved to be useful:

    ______________________________________                                        pyrazolo derivative                                                                            0.05 to 50 mmole/liter,                                                       preferably 0.1 to 1 mmole/liter                              coupler          0.05 to 100 mmole/liter                                      buffer, pH 5-10, 0.05 to 1 mole/liter, preferably                             preferably 6.5-8 0.1-0.5 mole/liter                                           wetting agent    0-1.0 mole/liter, preferably                                                  0.05-0.1 mole/liter                                          a) peroxidase    1.0-5000 KU/liter                                            b) hydrogen peroxide or                                                                        0.1-10 mmole/liter                                           hydrogen peroxide-                                                            producing substrate/                                                          enzyme mixture                                                                other adjuvants  0-5 mole/liter.                                              ______________________________________                                    

The above-given concentration ranges are to be so understood that thelower ranges are, in each case, preferred for photometric tests incuvettes and the upper ranges for rapid tests or tests on solidcarriers.

The pyrazolo derivatives useable according to the present inventiondisplay many advantages. Thus, because of the quantitative or almostquantitative reaction with the corresponding couplers, they makepossible a very high colour yield. The coloured materials produced bythe oxidative coupling and especially those which arise with anilinederivatives according to published European Patent Specification No.A-0,175,250 as couplers are stable and display a high extinction.Furthermore, the absorption maximum (λ_(max)) of the coloured materialsis so high that even haemolysed plasma or serum as sample material canbe used. The pyrazolo derivatives useable according to the presentinvention are also characterised by a rapid colour formation with acoupler under oxidative coupling conditions. Furthermore, the pyrazoloderivatives useable according to the present invention show only a lowtendency towards auto-oxidation so that they display the storagestability necessary for commercial tests.

The present invention also provides pyrazolo derivatives which are notknown from the prior art and thus are new and which have the generalformula: ##STR6## wherein X'--Y' signifies NR^(1') --CO or N═CR^(2'),whereby R^(1') is an alkyl radical and R^(2') is an alkyl, alkenyl,alkoxy, alkylthio, aryl, aralkyl radical optionally substituted byhydroxyl, dialkylphosphinyl, carboxyl, SO₃ H, PO₃ H₂, a salt of theseacid residues and/or alkoxycarbonyl; amino, which is optionallysubstituted by one or two alkyl radicals which, in turn, are optionallysubstituted by one or more hydroxyl, carboxyl and/or alkoxycarbonylradicals, whereby, when amino is substituted by two alkyl radicals,these radicals can be joined to form a ring which, apart from thenitrogen atom of the amino group, can also be interrupted by oxygen,sulphur or a further nitrogen atom or amino is optionally substituted byone or two acyl radicals, alkoxy and/or aralkoxycarbonyl radicals, H₂N--CO, alkyl, aralkyl- and/or arylcarbamoyl radicals; or hydrogen,carboxyl alkoxycarbonyl, carboxamido or halogen and Z' signifies NR^(3')--N═N, whereby R^(3') is an alkyl or aralkyl radical or Z' is anunsaturated chain with 3 to 5 members of nitrogen or of carbon andoptionally one or more nitrogen or sulphur atoms, whereby all carbonatoms of the unsaturated chain are parts of a double bond and wherebycarbon atoms are optionally substituted by alkyl, alkoxy, alkylthio,hydroxyl, aralkyl, aryl, carboxyl, carboxamido, alkoxycarbonyl, cyano,amino, which is optionally substituted by one or two alkyl radicalswhich, in turn, are optionally substituted by one or more hydroxyl,carboxyl and/or alkoxycarbonyl radicals, or halogen and whereby nitrogenatoms which are not bound via a double bond are substituted by alkyl oraralkyl or two neighbouring chain substituents optionally form analkylene radical which, in turn, is optionally substituted or anellatedwith aryl, as well as the corresponding tautomers and the salts thereof,with the proviso that

a) when X'--Y' is N═CR^(2') with R^(2') being alkyl or phenyl and Z' isan unsaturated chain of 4 carbon atoms, this is not unsubstituted and isnot substituted by alkyl, alkoxy or halogen,

b) when Z' is an unsaturated chain of 4 members with 3 carbon atoms andone nitrogen atom on the end of the chain so that in general formula(I') the ring with Z' forms a pyrimidine ring and one or more carbonatoms are substituted by alkyl, alkoxy, alkylthio, hydroxyl, aryl,alkoxycarbonyl or halogen and X'--Y' stands for N═CR^(2'), then R^(2')does not signify a hydrogen atom,

c) when Z' has the meaning: ##STR7## so that in general formula (I') thering with Z' forms a 1,2,4-triazine ring and X'--Y' stands forN═CR^(2'), then R^(2') is not a methyl radical:

The meanings of the individual substituents correspond to those givenfor general formula (I). Furthermore, the statements made for thepyrazolo derivatives useable according to the present invention withregard to advantageous radicals and groups apply in the same way for thenew pyrazolo derivatives.

A further subject of the present invention is a process for thepreparation of pyrazolo derivatives of general formula (I), wherein acompound of the general formula: ##STR8## in which X'--Y' and Z' havethe same meanings as in general formula (I') and R is a hydrogen atom,is converted

a) by reaction with nitric acid or nitric acid in admixture withsulphuric acid and/or acetic anhydride into a compound of generalformula (XIV) in which R is a nitro group; or

b) by reaction with nitrous acid into a compound of general formula(XIV) in which R is a nitroso group; or

c) by reaction with an aromatic diazonium salt into a compound ofgeneral formula (XIV) in which R is an arylazo radical,

and subsequently reduced.

The synthesis of the compounds of general formula (I') can take placeaccording to known methods by converting a compound of general formula(XIV) by known methods into the corresponding amino compound.

Nitro, nitroso and arylazo radicals, i.e. aryl-N═N-- radicals in whicharyl can have the same meanings as previously given for aryl radicalsand other groups containing such radicals, can be converted into aminogroups by reduction with reagents such as zinc in an acid, for examplehydrochloric acid, or glacial acetic acid sodium dithionite, tin in anacid, for example hydrochloric acid, stannous chloride, or bycatalytical hydrogenation, for example in the presence ofpalladium/carbon. Such reactions are described in Houben-Weyl, Methodender organischen Chemie, Vol. 11/1, pp. 341 et seq.

The introduction of nitro, nitroso or arylazo groups starting fromcompounds of general formula (XIV), in which R is a hydrogen atom, cantake place by nitration with nitric acid or nitric acid in admixturewith concentrated sulphuric acid or acetic anhydride.

By nitrosation with nitrous acid or by azo coupling with aromaticdiazonium salts, the nitroso group or an arylazo radical can beintroduced. Examples of such reactions are described in Houben-Weyl,Methoden der organischem Chemie, Vol. 10/1 and 10/3.

In some cases, the direct nitrosation of compounds of general formula(XIV) is possible in which R is an alkoxycarbonyl radical, for examplean acetyl radical, without first having to carry out a hydrolysis byboiling with concentrated hydrochloric acid to give a compound ofgeneral formula (XIV) in which R is a hydrogen atom (cf. Chem. Pharm.Bull., 22, 482/1974).

If heterocyclic compounds of general formula (XIV) are present in whichR is a carboxyl, alkoxycarbonyl or alkylcarbonyl radical, then these canbe converted into compounds of general formula (XIV), in which R is ahydrogen atom, by hydrolysis with concentrated hydrochloride acid or, inthe case of carboxylic acids, by thermal decarboxylation. This is thenfollowed by the introduction of a nitro, nitroso or arylazo radical.

Nitrogen atoms which are not on a double bond and in which radicalsX'--Y' and Z' occur with the structure given in general formula (I')must be alkylated or aralkylated. The N-alkylation or N-aralkylation canbe carried out by reaction of the appropriate compounds of generalformula (XIV), in which R has the above-given meaning, with alkylationor aralkylation agents, for example alkyl or aralkyl halides, dialkyl ordiaralkyl sulphates or arylsulphonic acid alkyl or aralkyl esters in thepresence of a base, for example sodium hydride, a tertiary amine, analkali metal carbonate or sodium hydroxide, in a solvent, for exampledimethylformamide or an aqueous-alcoholic system.

The starting materials necessary have either been described or can besynthesised analogously to known compounds. Information regarding thepreparation of the heterocyclic systems are contained in the followingpublications: G. P. Ellis, "Synthesis of fused Heterocycles", in "TheChemistry of Heterocyclic Compounds", E. C. Taylor, ed., 1987, pub. JohnWiley & Sons; P. N. Preston, "Condensed Imidazoles" in "The Chemistry ofHeterocyclic Compounds", A. Weissberger and E. C. Taylor, eds., 1986,John Wiley & Sons; Adv. of Het. Chem., 36, 343/1984; Chem. Pharm. Bull.,22, 482/1974; J. Het. Chem., 12, 481/1975; Chem. Pharm. Bull., 22,1814/1974; Ann., 660, 104/1962; Chem. Pharm. Bull., 23, 452/1975; J.Het. Chem., 10. 411/1973; and J. Chem. Soc. Perkin I, 2047/1977.

The compounds of general formula (I') are bases which form salts withorganic and inorganic acids which salts can advantageously be used forisolation and purification.

The following Examples are given for the purpose of illustrating thepresent invention:

EXAMPLE 1 3-Amino-2-methoxypyrazolo[1,5-a]pyridine hydrochloride##STR9##

1.1 1.48 g. 2-methoxy-pyrazolo[1,5-a]pyridine (Bull. Chem. Soc. Jap.,49, 1980/1976) are dissolved with ice-cooling in 20 ml. concentratednitric acid and mixed dropwise with 10.5 ml. fuming nitric acid. Thereaction mixture is stirred for 30 minutes in an ice-bath and for 1 hourat ambient temperature. The reaction solution is poured on to ice, theprecipitate obtained is filtered off with suction and washed with water.There is obtained 1 g. (52% of theory)2-methocy-3-nitropyrazolo[1,5-a]pyridine; m.p. 213°-216° C.

1.2 0.8 g. of the nitro compound obtained in 1.1 is suspended in 80 ml.2N hydrochloric acid and mixed with zinc dust while stirring vigorously.After repeated addition of zinc dust, after 1 hour a clear solution isobtained over a sediment of excess zinc dust. This is filtered off andthe filtrate is adjusted to pH 7 with sodium hydroxide. The reactionmixture is extracted with ethyl acetate. The organic phase is evaporatedand the remaining oil is filtered with ethanol over a short layer ofsilica gel. The eluate is evaporated, the residue is dissolved indiethyl ether and the ethereal solution is mixed with etherealhydrochloric acid. The precipitate obtained is filtered off with suctionand again recrystallised from isopropanol. There is obtained 0.33 g.(42% of theory) of the title compound; m.p. 238°-241° C.

TLC (silica gel, acetone/methylene chloride/glacial acetic acid 50:45:5v/v/v)=R_(f) =0.6.

EXAMPLE 2 3-Amino-2-chloro-5,7-dimethylpyrazolo[1,5-a]pyrimidinehydrochloride ##STR10##

Analogously to Example 1, starting from2-chloro-5,7-dimethylpyrazolo[1,5-a]pyrimidine (see Ann. Chem., 647,116/1961), there is obtained3-amino-2-chloro-5,7-dimethylpyrazolo[1,5-a]pyrimidine hydrochloride;m.p. 227°-230° C.

TLC (silica gel, acetone/methylene chloride/glacial acetic acid 50:45:5v/v/v): R_(f) =0.8.

EXAMPLE 3 3-Amino-2-methylpyrazolo[1,5-a]pyridine hydrochloride##STR11##

7 g. 3-Acetyl-2-methylpyrazolo[1,5-a]pyridine (see J. Org. Chem., 42,443/1977) are dissolved in 140 ml. 6N hydrochloric acid and mixeddropwise at 0° C. with a solution of 5.52 g. sodium nitrite in water.After 2 hours, the ice-bath used for the cooling is removed and thereaction mixture is left to stand overnight at ambient temperature andthen adjusted to pH 9. The precipitated nitroso compound (6.4 g.) isfiltered off with suction and dissolved in about 150 ml. 2N hydrochloricacid. The nitroso compound is reduced with zinc dust analogously toExample 1.2. The crude product is chromatographed on silica gel withethyl acetate. The product-containing fractions are evaporated, theresidue is dissolved in ethanol and the solution is mixed with ethanolichydrogen chloride. The precipitate which is obtained after some time isfiltered off with suction and dried. There are obtained 3.1 g. (45% oftheory) of the title compound; m.p. >275° C.; TLC (silica gel, ethylacetate/acetone/glacial acetic acid/water 50:25:12.5:12.5 v/v/v/v):R_(f) =0.6.

EXAMPLE 4 3-Amino-2-methylpyrazolo[3.2-b]thiazole hydrochloride##STR12##

Analogously to Example 3, from 3-acetyl-2-methylpyrazolo[3,2-b]thiazole(see Chem. Pharm. Bull., 22, 482/1974) there is obtained3-amino-2-methylpyrazolo[3,2-b]thiazole hydrochloride; m.p. 215°-218°C.; TLC (silica gel, methanol): R_(f) =0,65.

EXAMPLE 5 3-Amino-2-methoxy-5,7-dimethylpyrazolo[1,5-a]pyrimidinehydrochloride ##STR13##

1.04 g. 2-Methoxy-5,7-dimethylpyrazolo[1,5-a]pyrimidine (see Ann. Chem.,647, 116/1961) are nitrosated analogously to Example 3. There isobtained 1.1 g. (91% of theory) of the corresponding 3-nitroso compoundwhich is reduced with zinc dust analogously to Example 1.2. There isobtained 0.8 g. (80% of theory) of the title compound; m.p. 187°-190° C.

TLC (silica gel, acetone/methylene chloride/glacial acetic acid 50:45:5v/v/v): R_(f) =0.6.

EXAMPLE 6

Analogously to Example 5, there are obtained the compounds set out inthe following Table by nitrosation of the 3H-starting heterocycliccompound and reduction of the nitroso group.

The starting heterocyclic compound of Example 6b) is described in J.Het. Chem., 12, 481/1975, those of Examples 6c) and 6d) in J. Het.Chem., 18, 1149/1981. The 3H-starting heterocyclic compound of Example6a) is prepared analogously to the corresponding 2-methoxy compound inExample 1 by alkylation with ethyl bromoacetate (analogously to Bull.Chem. Soc. Jap., 49, 1980/1976).

                                      TABLE                                       __________________________________________________________________________    Example No.                                                                          structure            m.p.  R.sub.f.sup.1)                              __________________________________________________________________________    6a                                                                                    ##STR14##           179-184° C.                                                                  0.75.sup.2)                                 6b                                                                                    ##STR15##           >240° C.                                                                     0.75.sup.3)                                 6c                                                                                    ##STR16##           >250° C.                                                                     0.35.sup.4)                                 6d                                                                                    ##STR17##           205-213° C.                                                                  0.6.sup.5)                                  __________________________________________________________________________     .sup.1) TLC on silica gel                                                     .sup.2) elution agent: acetone/methylene chloride/glacial acetic acid         50:45:5 v/v/v                                                                 .sup.3) elution agent: methanol                                               .sup.4) elution agent: ethyl acetate/acetone/glacial acetic acid/water        50:25:12.5:12.5 v/v/v/v                                                       .sup.5) elution agent: ethyl acetate                                     

EXAMPLE 7 3-Amino-4-benzylpyrazolo[1,5-a]imidazole dihydrochloride##STR18##

0.4 g. 4-Benzyl-3-nitrosopyrazolo[1,5-a]imidazole (see Chem. Pharm.Bull., 22, 482/1974) are reduced with zinc in hydrochloric acidanalogously to Example 1.2 and converted with ethanolic hydrogenchloride into the dihydrochloride. There is obtained 0.37 g. (85% oftheory) 3-amino-4-benzylpyrazolo[1,5-a]imidazole dihydrochloride; m.p.158° C. (decomp.).

TLC (silica gel, acetone/methylene chloride/glacial acetic acid): R_(f)=0.4.

EXAMPLE 8 3-Amino-2-phenylpyrazolo[1,5-a]quinoline ##STR19##

1.0 g. 2-Phenylpyrazolo[1,5-a]quinoline (see J. Het. Chem., 12,481/1975) are nitrosated analogously to Example 3. There is obtained 1g. (89% of theory) of the corresponding 3-nitroso compound which issuspended in 90 ml. ethanol and mixed with 200 mg. palladium on carbon.The mixture is heated to 80° C. and 0.25 ml. hydrazine hydrate addedportionwise thereto. After 10 minutes, the palladium-carbon is filteredoff and the filtrate is evaporated. The residue is dissolved in ethanoland mixed with ethanolic hydrogen chloride. The precipitatedhydrochloride is filtered off with suction to give 0.7 g. (72% oftheory) of the title compound; m.p. 255°-258° C. TLC (Silicagel/methylene chloride): R_(f) =0.2

EXAMPLE 9 3-Amino-2,4-dimethylpyrazolo[1,5-a]imidazole dihydrochloride##STR20##

9.1 1.92 g. 1,2-Dimethylimidazole are dissolved in 10 ml. methylenechloride and mixed, while cooling with ice, with a solution ofO-p-toluenesulphonyl hydroxylamine which is obtained by the reaction of15.4 g. O-p-toluenesulphonylacethydroxamic acid ethyl ester with 118 ml.60% aqueous perchloric acid. The reaction mixture is stirred for 3 hoursat ambient temperature. The precipitate obtained is filtered off andwashed with diethyl ether. There are obtained 5 g. (88% of theory)N-amino-1,2-dimethylimidazolium-p-toluenesulphonate.

9.2 5 g. of the product obtained in 9.1 are heated with 5.4 g. sodiumacetate and 125 ml. acetic anhydride for 1 hour to 140° C. (bathtemperature). The reaction mixture is evaporated in a vacuum and theresidue is taken up in water. The pH value is adjusted to about 9 to 10and then followed by extraction with methylene chloride. The organicphase is dried and evaporated and the residue is chromatographed onsilica gel with ethyl acetate. There is obtained 0.45 g. (11% of theory)3-acetyl-2,4-dimethylpyrazolo[1,5-a]imidazole; m.p. 165°-167° C. 9.3 Thecompound obtained in 9.2 is nitrosated analogously to Example 3. Thereis obtained 0.35 g. (94% of theory) of the corresponding 3-nitrosocompound (m.p. 179°-183° C.) which is dissolved in 100 ml. diluteaqueous sodium bicarbonate solution and reduced with sodium dithionite.The reaction mixture is evaporated and the residue is digested withethanol. The ethanolic solution is evaporated and the product isprecipitated out by the addition of ethereal hydrochloric acid. There isobtained 0.3 g. (80% of theory) of the title compound; m.p. 199°-204° C.(decomp.).

TLC (silica gel, ethyl acetate/acetone/glacial acetic acid/water50:25:12,5:12,5 v/v/v/v): R_(f) =0.2.

EXAMPLE 10 3-Amino-2,4-dimethyl-6-phenylpyrazolo[3,2-c]-s-triazolehydrochloride ##STR21##

10.1 0.84 g. 2-Methyl-6-phenylpyrazolo[3,2-c]-s-triazole (see J. Chem.Soc. Perkin I, 2047/1977) are dissolved in 15 ml. dimethylformamide andmixed with 0.95 g. p-toluenesulphonic acid methyl ester. To this mixtureis added portionwise 0.2 g. of 55% sodium hydride, followed by stirringfor 1 hour at ambient temperature. The reaction mixture is poured on toice and the mixture is extracted with ethyl acetate. There is obtained 1g. of crude 2,4-dimethyl-6-phenylpyrazolo[3,2-c]-s-triazole.

TLC (silica gel, methylene chloride/ethyl acetate 50:1 v/v): R_(f) =0.5.

10.2 To a solution of 1.8 g.2,4-dimethyl-6-phenylpyrazolo[3,2-c]-s-triazole and 0.5 g. sodiumacetate in 8 ml. glacial acetic acid is added portionwise phenyldiazonium tetrafluoroborate which was obtained by diazotisation of 2.2ml. aniline. The reaction mixture is left to stand overnight, thendiluted with water and the orange-yellow precipitate is filtered offwith suction. The 3-phenylazo compound is purified by chromatography onsilica gel with methylene chloride. Yield 1.3 g. (73% of theory); m.p.183°-186° C.

10.3 1.3 g. 2,4-Dimethyl-6-phenyl-3-(phenylazopyrazolo[3,2-c]-s-triazolefrom 10.2 is suspended in a mixture of 100 ml. ethanol and 100 ml.diluted aqueous sodium bicarbonate solution and mixed with an excess ofsodium dithionite. The reaction mixture is stirred for about 2 days at60° C. During this time, further sodium dithionite, sodium bicarbonateand ethanol (1 litre) are added thereto. The reaction mixture isevaporated and the residue is taken up in ethyl acetate. The organicphase is thoroughly washed with water, dried and evaporated. The residueis dissolved in ethanol and the hydrochloride is precipitated out by theaddition of ethanolic hydrogen chloride. There is obtained 0.8 g. of thetitle compound; m.p. 210°-213° C.

TLC (silica gel, ethyl acetate/acetone/glacial acetic acid/water50:25:12.5:12.5 v/v/v/v): R_(f) =0.7.

EXAMPLE 11 3-Amino-pyrazolo[1,5-b]pyridazine hydrochloride ##STR22##

11.1 1.8 g. Pyrazolo[1,5-b]pyridazine-3-carboxylic acid ethyl ester (seeChem. Pharm. Bull., 22, 1814/1974) are boiled under reflux for 5 hourswith 36 ml. 6N hydrochloric acid. The reaction mixture is evaporated andthe residue is dissolved in water. The solution is neutralised withsodium carbonate and extracted three times with ethyl acetate. Theorganic phase is dried and evaporated. There is obtained 0.85 g. (75% oftheory) of oily pyrazolo[1,5-b]pyridazine.

TLC (silica gel, methylene chloride/ethyl acetate 2:1 v/v): R_(f) =0.6.

11.2 0.8 g. of the compound obtained in 11.1 is nitrosated analogouslyto Example 8. There is obtained 0.6 g. (60% of theory)3-nitrosopyrazolo[1,5-b]pyridazine; m.p. 129°-132° C.

The nitroso compound is reduced with palladium/hydrazine analogously toExample 8. There is obtained 0.4 g. (58% of theory) of the titlecompound; m.p. 230° C. (decomp.).

TLC (silica gel, ethyl acetate/methanol 1:1 v/v): R_(f) =0.6.

EXAMPLE 12 3-Amino-2,6-dimethylpyrazolo[1,5-a]pyrimidine hydrochloride##STR23##

3.2 g. 2,6-Dimethyl-3-nitropyrazolo[1,5-a]pyrimidine (see Synthesis,673/1982) are dissolved in 320 ml. ethanol and mixed with 320 ml. of a5% aqueous solution of sodium bicarbonate. 14.2 g. Sodium dithionite arenow added to this mixture portionwise with stirring and cooling untilthe thin layer chromatogram shows the absence of starting material. Thereaction mixture is concentrated somewhat and extracted three times withethyl acetate. The organic phase is dried and evaporated. The residue isdissolved in a little ethanol and mixed with an equimolar amount ofhydrogen chloride in diethyl ether. The precipitated crystals arefiltered off with suction. There are obtained 2.9 g. (80% of theory) ofthe title compound; m.p. 224° C. (decomp.).

TLC (silica gel, chloroform/methanol/methyl ethyl ketone/glacial aceticacid/water 75:35:25:5:8 v/v/v/v/v): R_(f) =0.73.

EXAMPLE 133-Amino-4-methyl-6-methylthio-2-phenylpyrazolo[3,2-c]-s-triazolehydrochloride ##STR24##

13.1 4.6 g. 6-Methylthio-2-phenylpyrazolo[3,2-c]-s-triazole (see J.Chem. Soc. Perkin I, 2047/1977) are dissolved in 46 ml. drydimethylformamide and mixed with 4.4 g. p-toluenesulphonic acid methylester. 1 g. of approximately 55% sodium hydride are added portionwisethereto with stirring. The reaction mixture is stirred for 1 hour atambient temperature and then poured on to ice/water. Extraction iscarried out with ethyl acetate and the organic phase is dried andevaporated. The crude product is purified by chromatography on silicagel with methylene chloride/ethyl acetate (50:1 v/v). There is obtained1.5 g. (30% of theory) of the N-4-methylated heterocyclic compound.

TLC (silica gel, methylene chloride/ethyl acetate 50:1 v/v): R_(f)=0.35.

13.2 1.2 g. of the compound obtained in 13.1 is dissolved in 36 ml.glacial acetic acid and mixed with 1.33 g. 4-methoxyphenyldiazoniumtetrafluoroborate. After 2 hours, the same amount of the diazonium saltis again added thereto and the reaction mixture is stirred overnight.The reaction mixture is diluted with water and the pH value is adjustedto 5 with a dilute aqueous solution of sodium hydroxide. The precipitateobtained is filtered off, washed with water and dried in a vacuum at 50°C. There is obtained 1.9 g. (100% of theory) of the corresponding 3-azocompound.

13.3 The azo compound obtained in 13.2 is suspended in 100 ml. glacialacetic acid and reduced by the addition of zinc dust. The excess zinc isfiltered off and the reaction mixture is evaporated. For purification,the crude product is dissolved in 100 ml. dioxan and mixed with 1.5 g.di-tert.-butyl dicarbonate. The mixture is stirred for 2 hours,evaporated and the product obtained is chromatographed on silica gelwith methylene chloride/ethyl acetate (100:0 to 90:10 v/v). Thefractions which contain the desired N-tert.-butoxycarbonylated aminocompound are combined, evaporated and the residue is suspended in 50 ml.ethanol. For the splitting off of the tert.-butoxycarbonyl radical, 50ml. ethanolic hydrochloric acid are added thereto, followed by stirringat ambient temperature. The reaction mixture is evaporated to dryness,dissolved in hot ethanol, filtered and the filtrate is mixed withdiethyl ether. The precipitate obtained is filtered off with suction.There is obtained 0.6 g. (40% of theory) of the title compound; m.p.207°-210° C.

TLC (silica gel, ethyl acetate): R_(f) =0.65.

EXAMPLE 14 3-Amino-4-chloro-2-methoxypyrazolo[1,5-a]pyridinehydrochloride ##STR25##

7.5 g. N-Amino-3-chloropyridinium mesitylene-sulphonate (see J. Chem.Soc. Perkin I, 2580/1973) are dissolved in 50 ml. dry dimethylformamideand mixed with 3.5 g. finely powdered potassium carbonate. 28 ml. of a5M diketene solution in methylene chloride are added thereto withcooling. The reaction mixture is stirred for 30 minutes at 0° C. and for2 hours at ambient temperature. The precipitate obtained is filtered offwith suction, the filtrate is evaporated and the residue obtained ispurified by chromatography on silica gel with methylene chloride/5-10%methanol. There are obtained 2.7 g. of the N-acetoacetylated compoundwhich is dissolved in 100 ml. ethanol and mixed with 2.6 g. of finelypowdered potassium carbonate. The reaction mixture is stirred for 2 daysat ambient temperature and finally evaporated. The residue is dissolvedin 100 ml. dry dimethylformamide, the solution is cooled and mixed with7 g. methyl iodide. The reaction mixture is stirred for 1 hour at 0° C.and for 30 hours at ambient temperature. The dimethylformamide isdistilled off and the residue is dissolved in water/ethyl acetate. Theethyl acetate phase is separated off, dried and evaporated. The productobtained is purified by column chromatography on silica gel withmethylene chloride/ethyl acetate (20:1 v/v). There is obtained 0.8 g.3-acetyl-4-chloro-2-methoxypyrazolo[1,5-a]-pyridine which is nitrosatedand reduced analogously to Example 3. Yield: 0.3 g. of the titlecompound; m.p. 200°-230° C. (decomp.).

TLC (silica gel, methylene chloride/ethyl acetate 1:1 v/v): R_(f) =0.6.

EXAMPLE 153-Amino-2-methyl-5,6,7,8-tetrahydropyrazolo[3,2-b]-benzothiazolehydrochloride ##STR26##

15.1 28.5 g. Acetamide and 21.3 g. phosphorus pentasulphide aresuspended in 32 ml. anhydrous toluene and mixed, while stirring, with 5ml. of a mixture of 85 g. 2-bromocyclohexanone and 40 ml. anhydroustoluene. The reaction mixture is heated to 75° to 80° C. and theremainder of the bromoketone solution is added thereto. The reactionmixture is boiled under reflux for 10 minutes, cooled and adjusted to pH8 to 9 with a 2N aqueous solution of sodium hydroxide. Extraction iscarried out with ethyl acetate, the organic phase is washed with water,dried and evaporated. The residue is distilled in a vacuum to give 14.4g. 2-methyl-4,5,6,7-tetrahydrobenzothiazole. b.p. 62° C./0.5 mm. Hg.

15.2 2.06 g. 2-Methyl-4,5,6,7-tetrahydrobenzothiazole are dissolved inabout 10 ml. methylene chloride, the mixture is cooled and mixed with 30mMole of a methylene chloride solution ofO-p-toluenesulphonyl-hydroxylamine. The reaction mixture is stirred for2 hours on an ice-bath and the product is precipitated out by theaddition of diethyl ether. There are obtained 6.4 g. (94% of theory)N-amino-2-methyl-4,5,6,7-tetrahydrobenzothiazolium-p-toluenesulphonate;m.p. 127°-140° C.

15.3 6.2 g. of the N-amino compound obtained in 15.2 are, together with4.7 g. sodium acetate and 16 ml. acetic anhydride, heated to 140° C.(bath temperature). After 1 hour, the mixture is evaporated, the residueis dissolved in water/methylene chloride, rendered alkaline with sodiumcarbonate and extracted three times with methylene chloride. The productobtained is purified by column chromatography on silica gel with ethylacetate/ligroin (1:2 v/v). There are obtained 2.1 g. (50% of theory)3-acetyl-2-methyl-5,6,7,8-tetrahydropyrazolo[3,2-b]benzothiazole; m.p.90°-92° C.

15.4 1.4 g. of the compound obtained in 15.3 is nitrosated analogouslyto Example 3 and the nitroso compound is reduced. There is obtained 1.3g. of the title compound; m.p. 239°-240° C. (decomp.).

TLC (silica gel, ethyl acetate): R_(f) =0.5.

EXAMPLE 16 3-Amino-2,5-dimethyl-6-phenylpyrazolo[3,2-]thiazolehydrochloride ##STR27##

Analogously to Example 15, starting from α-bromopropiophenone, there isobtained the title compound; m.p. 252°-254° C. (decomp.).

TLC (silica gel, ethyl acetate): R_(f) =0.6

EXAMPLE 17 3-Amino-pyrazolo[1,5-a]pyridine hydrochloride ##STR28##

17.1 8.2 g. Pyrazolo[1,5-a]pyridine (see Ann. Chem., 498/1977) aredissolved in 30 ml. 6N hydrochloric acid, the solution is cooled to 0°C. and a solution of 6.9 g. sodium nitrite in 30 ml. water slowly addeddropwise thereto. After 1 hour, the nitrosation is complete. About 100ml. water are added thereto followed by repeated extraction with ethylacetate. The organic phase is dried and evaporated. There are obtained9.6 g. 3-nitrosopyrazolo[1,5-a]pyridine.

17.2 9 g. of the above-obtained nitroso compound are introduced into asolution of 22 g. stannous chloride dihydrate in 180 ml. concentratedhydrochloric acid. The reaction mixture is stirred for 1 hour at ambienttemperature and, for the completion of the reduction, mixed with 8 g.stannous chloride dihydrate in 30 ml. concentrated hydrochloric acid.The suspension is poured on to about 150 g. ice, adjusted with sodiumhydroxide to pH 12 and quickly extracted with ethyl acetate. The ethylacetate phase is dried and evaporated. The residue obtained is dissolvedin about 350 ml. diethyl ether and mixed with ethereal hydrochloricacid. The precipitate obtained is filtered off, washed with diethylether and dried. There are obtained 11.3 g. (100% of theory) of thetitle compound; m.p. 228°-232° C.

TLC (silica gel, ethyl acetate/methanol 9:1 v/v): R_(f) =0.52.

EXAMPLE 18 3-Amino-2-methylthiopyrazolo[1,5-a]pyridine hydrochloride.##STR29##

4 g. 2-Methylthio-3-nitropyrazolo[1,5-a]pyridine (see Heterocycles, 6,379/1977; Chem. Pharm. Bull., 25, 1528/1977) are dissolved in 200 ml.concentrated hydrochloric acid and mixed with 20 g. stannous chloridedihydrate. After 1 hour, there are again added 15 g. stannous chloridedihydrate, 15 ml. concentrated hydrochloric acid and 200 ml. water.After a further reaction period of 1 hour, the reaction mixture ispoured on to ice, the yellow solution is rendered alkaline and extractedwith ethyl acetate. The organic phase is dried and evaporated, theresidue is dissolved in a little ethanol and mixed with etherealhydrochloric acid in order to form the hydrochloride. There are obtained3.9 g. (95% of theory) of the title compound; m.p. 226° C.

TLC (silica gel, methylene chloride/tert.-butyl methyl ether 2:8 v/v):R_(f) =0.6.

EXAMPLE 19 R,S-3-Amino-2-(1-hydroxyethyl)-pyrazolo[1,5-a]pyridinehydrochloride ##STR30##

25 g. N-aminopyridine hydroiodide are dissolved in 250 ml. drydimethylformamide and mixed, while stirring, with 17.5 g. potassiumcarbonate. Subsequently, while stirring, 20.5 g.R,S-2-hydroxy-5-oxobut-3-yne (see J. Chem. Soc. Perkin I, 1908/1972) areadded dropwise thereto. The reaction mixture thereby warms up and isleft to stand overnight. After the addition of 1.25 liters of water, thereaction mixture is extracted several times with ethyl acetate. Thecombined extracts are dried and evaporated. The remaining oil is dilutedwith some diethyl ether. After some time, precipitated crystals arefiltered off with suction. There are obtained 9.8 g. (42% of theory)R,S-3-acetyl-2-(1-hydroxyethyl)-pyrazolo[1,5-a]pyridine (R_(f) (silicagel, methylene chloride/ethyl acetate 1:1 v/v)=0.35) which is nitrosatedanalogously to Example 3. The nitroso compound is reduced analogously toExample 8 with palladium-carbon/hyrazine hydrate. There are obtained 6.8g. of the title compound, the crystals of which contain 1.8 molehydrogen chloride and melt at 229°-231° C. (decomp.). R_(f) (silica gel,ethyl acetate/methanol 3:1 v/v)=0.65.

EXAMPLE 203-Amino-2,5-dimethyl-7-(dimethylamino)-pyrazolo[1,5-a]pyrimidinehydrochloride ##STR31##

20.1 1.6 g. 2,5-Dimethyl-7-hydroxypyrazolo[1,5-a]pyrimidine are heatedunder reflux for 40 minutes with 16.6 ml. phosphorus oxychloride and 0.8ml. N,N-dimethylaniline. Excess phosphorus oxychloride is distilled offand the residue is poured on to ice. Extraction is carried out withmethylene chloride, the organic phase is washed with an aqueous solutionof sodium carbonate, dried and evaporated. The residue obtained isdissolved in 28 ml. ethanol and mixed with 2 g. of a 40% solution ofdimethylamine in water. The mixture is stirred for 2.5 hours at ambienttemperature, evaporated and the residue chromatographed over silica gelwith ethyl acetate. There is obtained 0.86 g. (46% of theory)2,5-dimethyl-7-(N,N-dimethylamino)pyrazolo[1,5-a]pyrimidine which isnitrosated analogously to Example 3. The nitroso compound is reducedanalogously to Example 8 with palladium-carbon/hydrazine hydrate. Thereis obtained 0.8 g. of the title compound. R_(f) (silica gel,chloroform/methanol/methyl ethyl ketone/glacial acetic acid/water75:35:25:5:8 v/v/v/v/v)-0.54.

EXAMPLE 21 3-Amino-4-methylpyrazolo[1,5-a]imidazole hydrochloride.##STR32##

21.1 Analogously to the process described in "Organikum" (pub. VEBDeutscher Verlag der Wissenschaften, Berlin, pp. 514-515), 65 g. ethylethoxymethylenecyanoacetate are reacted with 55 g.2,2-diethoxyethylhydrazine to give 100.2 g.5-amino-1-(2,2-diethoxyethyl)-pyrazole-4-carboxylic acid which isdissolved in 4 litres ethanol and mixed with 2 litres 20% sulphuricacid. The reaction mixture is heated under reflux for 3 hours andneutralised by the addition of solid sodium bicarbonate. Precipitatedsalt is filtered off and the filtrate is evaporated. The residue isextracted several times with boiling methylene chloride and the extractsare combined and evaporated to give 58.2 g. (86% of theory)pyrazolo[1,5-a]imidazole-3-carboxylic acid ethyl ester; m.p. 126°-127°C. R_(f) (silica gel, ethyl acetate)=0.64.

21.2 18.7 g. of the above-obtained carboxylic acid ester are dissolvedin 190 ml. dimethylformamide and mixed with 17 g. p-toluenesulphonicacid methyl ester. While stirring, 3.98 g. 55% sodium hydride areintroduced portionwise. The reaction mixture is stirred for 30 minutesat ambient temperature, then poured on to ice/water and extracted withethyl acetate. There are obtained 21.9 g. (100% of theory)4-methylpyrazolo[1,5-a]imidazole-3-carboxylic acid ethyl ester which issaponified by boiling with 600 ml. concentrated hydrochloric acid. Theresultant carboxylic acid is simultaneously decarboxylated to give 16.7g. (83% of theory) 4-methylpyrazolo[1,5-a]imidazole dihydrochloride(R_(f) =0.39, silica gel, methylene chloride with 5% by volume ofmethanol).

21.3 12.8. g. Aniline are diazotised in a solution of 12.8 ml.concentrated sulphuric acid in 64 ml. water by the addition of asolution of 9.5 g. sodium nitrite in 42 ml. water. The solution isadjusted to pH 5 by the addition of sodium hydroxide and mixed dropwiseat 5° to 10° C. with a solution of 16.5 g. of the above-obtained4-methylpyrazolo[1,5-a]imidazole dihydrochloride in 165 ml. water and 14ml. glacial acetic acid. The reaction mixture is stirred for 1 hour at5° to 10° C. and then for 3 hours at ambient temperature, whereafter theseparated precipitate is filtered off with suction. There are obtained12 g. 4-methyl-3-phenylazopyrazolo[1,5-a]imidazole which is reduced withsodium dithionite analogously to Example 10.3 and purified analogouslyto Example 13.3. There are obtained 3.3 g.3-amino-4-methylpyrazolo[1,5-a]imidazole hydrochloride; m.p. 210° C.(decomp.).

R_(f) =0.23 (silica gel, ethyl acetate/acetone/glacial acetic acid/water50:25:12.5:12.5).

EXAMPLE 22 3-Amino-2,4,6-trimethylpyrazolo[3,2-c]-s-triazolehydrochloride ##STR33##

22.1 Starting from 4-ethoxycarbonyl-3-methylpyrazol-5-yl hydrazine (seeChem. Ber., 89, 2552/1956), there is prepared, in the manner describedin Example 6 of published Federal Republic of Germany PatentSpecification No. 18 10 462,2,6-dimethyl-4H-pyrazolo[3,2-c]-s-triazole-3-carboxylic acid ethylester, which, before acidic saponification, is methylated analogously toExample 10.1. As intermediate, there is thus obtained2,4,6-trimethylpyrazolo[3,2-c]-s-triazole-3-carboxylic acid ethyl ester(R_(f) =0.52; silica gel, ethyl acetate/ligroin 1:1 v/v) which issaponified by boiling with concentrated hydrochloric acid andsimultaneously decarboxylated. There is obtained2,4,6-trimethylpyrazolo[3,2-c]-s-triazole (R_(f) =0.24; silica gel,ethyl acetate/ligroin 1:1 v/v) as a pale yellowish oil whichcrystallises after some time.

22.2 Analogously to Example 13.2 and 13.3, the above-obtained product isreacted with p-methoxybenzene-diazonium salt, reduced and the productobtained purified. The title compound is obtained; m.p. 240° C.

R_(f) =0.50 (silica gel, ethyl acetate/acetone/glacial acetic acid/water50:25:12.5:12.5 v/v/v/v).

EXAMPLE 23 2-Acetamido-30amino-pyrazolo[1,5-a]pyridine hydrochloride.##STR34##

4 g. 2-Acetamidopyrazolo[1,5-a]pyridine (see Chem. Pharm. Bull., 21,2146/1973) are nitrosated analogously to Example 3. The nitroso compoundobtained is reduced with palladium-carbon/hydrazine hydrate analogouslyto Example 8. There are obtained 3.9 g. (67% of theory)2-acetamido-3-aminopyrazolo[1,5-a]pyridine hydrochloride; m.p. 255°-259°C. (decomp.).

R_(f) =0.5 (silica gel, ethyl acetate/acetone/glacial acetic acid/water50:25:12.5:12.5 v/v/v/v).

EXAMPLE 24 2-Vinyl-3-aminopyrazolo[1,5-a]pyridine hydrochloride##STR35##

25 g. R,S-3-Acetyl-2-(1-hydroxyethyl)-pyrazolo[1,5-a]pyridine (fromExample 22) are mixed with 50 ml. concentrated sulphuric acid and heatedfor 2 hours at a bath temperature of 95° C. The reaction mixture ispoured on to a large quantity of ice, rendered alkaline with sodiumhydroxide and extracted with ethyl acetate. The crude product obtainedis chromatographed over silica gel with ligroin/ethyl acetate (95:5 to90:10 v/v). There are obtained 3.4 g. 2-vinylpyrazolo[1,5-a]pyridine(R_(f) =0.6; silica gel, ligroin/ethyl acetate 3:1 v/v), which isreacted with phenyldiazonium salt analogously to Example 21.2, reducedand purified. The title compound is obtained. R_(f) =0.65; silica gel,ligroin/acetone/glacial acetic acid 50:45:5 v/v/v).

EXAMPLE 25 6-(3-Acetoxypropyl)-3-amino-2-methylpyrazolo[1,5-a]pyrimidine##STR36##

By the reduction of6-(3-acetoxypropyl)-2-methyl-3-nitropyrazolo[1,5-a]pyrimidine (seeSynthesis, p. 673/1982) analogously to Example 12, there is obtained thetitle compound. R_(f) =0.21 (silica gel, methylene chloride/methanol90:1 v/v).

EXAMPLE 26 2,3-Diaminopyrazolo[1,5-a]pyridine dihydrochloride ##STR37##

2.66 g. 2-aminopyrazolo[1,5-a]pyridine (see Chem. Pharm. Bull., 21,2146/1973) are reacted with p-methoxybenzenediazonium salt analogouslyto Example 13.2. The resultant azo compound is reduced with sodiumdithionite analogously to Example 10.3 and the crude product obtained ispurified analogously to Example 13.3. There is obtained the titlecompound; m.p. 190° C.

R_(f) =0.6 (silica gel, ligroin/acetone/glacial acetic acid 60:40:1v/v/v).

EXAMPLE 27 3-Aminopyrazolo[3,2-b]thiazole hydrochloride ##STR38##

27.1 60 g. of the potassium salt of dithiocarbazinic acid are reactedwith 60 ml. bromoacetaldehyde diethyl acetal in 400 ml.dimethylformamide at a bath temperature of 50° C. for 12 hours. Thereaction mixture is evaporated, the remaining emulsion is mixed with 500ml. water and the product is extracted with diethyl ether. The crudeproduct is purified by chromatography over silica gel (diethylether/ligroin 6:4 v/v). There are obtained 20.9 g. of thedithiocarbazinic acid 2,2-diethoxyethyl ester. 27.2 17.75 g. of theabove-obtained carbazinic acid ester and 11.3 g. formyl chloroaceticester are dissolved in 250 ml. ethanol and heated for 2 hours at 50° C.The reaction mixture is evaporated, the residue is mixed with water andthe product is extracted with diethyl ether. The ethereal phase is driedand evaporated. The residue is heated under reflux for 4 hours in 300ml. ethanolic hydrochloric acid. The reaction mixture is evaporated, theresidue is taken up in water and the reaction product is extracted withethyl acetate. For purification, it is chromatographed over silica gelwith ethyl acetate/ligroin (1:1 v/v). There are obtained 6.5 g.pyrazolo[3,2-c]thiazole-3-carboxylic acid ethyl ester.

R_(f) =0.65 (silica gel, toluene/dioxan/glacial acetic acid 72:20:8v/v/v).

27.3 5.8 g. of the above-obtained ester are heated under reflux for 7hours in a mixture of 300 ml. 3N hydrochloric acid and 15 ml. dioxan,whereby, every 2 hours, in each case 15 ml. concentrated hydrochloricacid are added thereto. The reaction mixture is adjusted to pH 8 by theaddition of sodium hydroxide and extracted several times with ethylacetate. After drying and evaporating the organic phase, there areobtained 3 g. pyrazolo[3,2-c]thiazole in the form of a yellowish oil.This product is nitrosated analogously to Example 3 and reduced. Thereare obtained 3.4 g. of the title compound as dihydrochloride; m.p. 170°C. (decomp.).

R_(f) =0,66 (silica gel, ethyl acetate/acetone/glacial acetic acid/water50:25:12.5:12.5 v/v/v/v).

EXAMPLE 28 3-Amino-4,6-dimethylpyrazolo[3,2-c]-s-triazole hydrochloride##STR39##

28.1 24.4 g. 3-Aminopyrazole-4-carboxylic acid ethyl ester (seeOrganikum, p. 514, pub. VEB Verlag der Wissenschaften, Berlin) aresuspended in 100 ml. concentrated hydrochloric acid and diazotised at 0°to 5° C. by the addition of a solution of 10.35 g. sodium nitrite in 50ml. water. Thereafter, a solution of 100 g. stannous chloride in 100 ml.concentrated hydrochloric acid is added thereto and the reaction mixtureis stirred for 2 hours in an ice-bath. The yellow precipitate obtainedis filtered off, dissolved in 150 ml. water and mixed with 20 ml. aceticanhydride. The reaction mixture is heated to a bath temperature of 80°C. and mixed portionwise over the course of 2 hours with, in all, 25 g.sodium bicarbonate. The neutral solution is then continuously extractedwith ethyl acetate for 24 hours. After drying and evaporating theorganic phase, there are obtained 17 g.3-acethydrazinopyrazole-4-carboxylic acid ethyl ester (R_(f) =0.33;silica gel, acetone/methylene chloride/glacial acetic acid 50:45:5v/v/v) which is cyclised with phosphorus oxychloride analogously to theprocedure described in Example 6 of published Federal Republic ofGermany Patent Specification No. 18 10 462. There are obtained 4.8 g.(32% of theory) 6-methylpyrazolo[3,2-c]-s-triazole-3-carboxylic acidethyl ester (R_(f) =0.70; silica gel, toluene/ethyl acetate/methanol2:1:1 v/v/v). 28.2 Analogously to Example 10.1, the above-obtainedheterocyclic compound is methylated and the product purified bychromatography over silica gel with ethyl acetate/ligroin (2:1 v/v). Byheating for 6 hours with 6N hydrochloric acid, the ester is saponifiedand the carboxylic acid formed as intermediate is decarboxylated. Theacid is neutralised by the addition of sodium carbonate and the productis extracted with ethyl acetate. There is obtained 1.1 g. of oily4,6-dimethylpyrazolo[3,2-c]-s-triazole (R_(f) =0.26; silica gel, ethylacetate/ligroin 1:1 v/v).

28.3 The above-obtained heterocyclic compound is nitrosated analogouslyto Example 3 and the nitroso group is reduced withpalladium-carbon/hydrazine analogously to Example 8. The title compoundis obtained as hydrochloride; m.p. 190° C. (decomp.).

R_(f) =0.53 (silica gel; ethyl acetate/acetone/glacial acetic acid/water50:25:12.5:12.5 v/v/v/v).

EXAMPLE 29 3-Amino-2-hydroxymethylpyrazolo[1,5-a]pyridine hydrochloride##STR40##

29.1. 10.5 g. Pyrazolo[1,5-a]pyridine-2-carboxylic acid ethyl ester (seeJ. Het. Chem., 18. 1149/1981) are dissolved in tetrahydrofuran and addedto a suspension of 5 g. sodium aluminium hydride in dry tetrahydrofuran.The reaction mixture is heated under reflux for 3 hours and then pouredinto a mixture of ice and methanol. The mixture is mixed with 100 ml.10% ammonium chloride solution and extracted with ethyl acetate. Theorganic phase is dried and evaporated. The residue is purified bychromatography on silica gel; with ligroin/ethyl acetate. There areobtained 7.3 g. 2-hydroxymethylpyrazolo[1,5-a]pyridine in the form of ayellow oil (R_(f) =0.49, silica gel, ethyl acetate).

29.2 The above-obtained compound is nitrosated analogously to Example 3and reduced with palladium-carbon/hydrazine hydrate analogously toExample 8. There is obtained the title compound; m.p. 218° C. R_(f)=0.62 (silica gel, ethyl acetate/methanol 2:1 v/v).

EXAMPLE 30 3-Amino-2-chloropyrazolo[1,5-a]pyridine hydrochloride##STR41##

2-Aminopyrazolo[1,4-a]pyridine (see Chem. Parm. Bull., 21, 2146/1973) isreacted with p-methoxybenzene diazonium salt analogously to Example13.2. 0.53 g of the 3-azo compound obtained is suspended in 10 ml. 6Nhydrochloric acid and mixed at +5° C., while stirring, with a solutionof 104 mg. sodium nitrite in 0.2 ml. water. After 30 minutes, a coldsolution of 198 mg. cuprous chloride in 6 ml. 6N hydrochloric acid isadded thereto and the reaction mixture is stirred for 40 hours atambient temperature. The mixture is mixed with water and extracted threeor four times with ethyl acetate. After purification of the crudeproduct by chromatography over silica gel (elution agent: ethylacetate/ligroin 1:1 v/v), there are obtained 320 mg. of2-chloro-3-(p-methoxyphenylazo)pyrazolo[1,5-a]pyridine (R_(f) =0.65;silica gel, ethyl acetate/ligroin 1:1 v/v) which is reduced with sodiumdithionite analogously to Example 10.3. There is obtained the titlecompound; m.p. 249°-251° C. (decomp.). R_(f) =0.2 (silica gel, ethylacetate/ligroin 1:1 v/v).

EXAMPLE 31

Analogously to Example 17, starting from the indicated substitutedpyridines, there are obtained the compounds shown in the followingTable:

    __________________________________________________________________________    starting material                                                                           product           m.p.                                                                              R.sub.f                                   __________________________________________________________________________    31.1                                                                              ##STR42##                                                                                ##STR43##        208° C.                                                                    0.18.sup.1)                               31.2                                                                              ##STR44##                                                                                ##STR45##        212° C.                                                                    0.23.sup.1)                               __________________________________________________________________________     .sup.1) ethyl acetate/methanol (9:1 v/v)                                 

EXAMPLE 32 3-Amino-2-morpholinopyrazolo[1,5-a]pyridine hydrochloride##STR46##

2-Aminopyrazolo[1,5-a]pyridine (see Chem. Pharm. Bull., 21, 2146/1973)is reacted with p-methoxybenzenediazonium salt analogously to Example13.2. 4 g. of the azo compound obtained are dissolved in 100 ml.dimethylformamide and mixed with 5.33 g. β,β'-dibromodiethyl ether and 2g. 55% sodium hydride. The reaction mixture is stirred for 1.5 hours atambient temperature and water and ethyl acetate added thereto. Theorganic phase is separated off and the aqueous phase is again extractedwith ethyl acetate. The combined organic phases are dried andevaporated. The residue is triturated with hexane. There are obtained4.75 g. 3-(4-methoxyphenyl)azo-2-morpholinopyrazolo[1,5-a]pyridine(R_(f) =0,7; silica gel, ethyl acetate/methylene chloride 1:1 v/v).

The azo compound is reduced with zinc in glacial acetic acid analogouslyto Example 13.3, purified and the tert.-butoxycarbonyl radical is splitoff with hydrochloric acid in ethanol. There are obtained 2.43 g. of thetitle compound; m.p. 105°-110° C. (decomp.). R_(f) =0.4 (silica gel,ethyl acetate).

EXAMPLE 33 Determination of hydrogen peroxide in aqueous solution

Reagent solutions:

Solution A

12 mg. peroxidase (=3000 U)

1 ml. 0.1N phosphate buffer, pH 8

Solution B

53.6 mg. 2,4,6-tribromohydroxybenzoic acid

1 ml. 0.1N phosphate buffer, pH 8

Solution C

32.1 mg. N-methyl-N-phenylaminomethanephosphonic acid hydrochloride(prepared according to published European Patent Specification No.A-0,175,250

1 ml. 0.1N phosphate buffer, pH 8

4.8×10⁻⁷ mole of a pyrazolo derivative useable according to the presentinvention are dissolved in about 5 to 9 ml. 0.1N phosphate buffer (pH8), possibly with the help of a little methanol, acetone ordimethylformamide. To this are added 50 μl. of Solution A and, dependingupon the desired coupler, either 200 μl. of Solution B or C. Finally,there are added thereto also 20 μl. of a 0.012 molar solution ofhydrogen peroxide in water, then made up with buffer to 10 ml. and thesolutions well mixed up. The solutions are measured in 1 cm. cuvettes.In the following Table 1, there are set out the wavelengths and theextinction at λ_(max) of the resultant coloured materials.

                  TABLE 1                                                         ______________________________________                                                   coupling component coupling component                              substance  TBHB.sup.1)        aniline.sup.2)                                  from       λ.sub.max   λ.sub.max                                Example No.                                                                              [nm]   ext.sup.3)  [nm] ext..sup.3)                                ______________________________________                                        1          568    69300       620  78200                                      2          536    18000       578  12600                                      3          622    28900       657  34800                                      4          572    30400       613  35600                                      5          557    36300       587  41900                                      6.1        568    39300       615  48800                                      6.2        628    19400       658  23000                                      6.3        602    22200       634  32600                                      6.4        587     7600       628  13300                                      7          626    30100       674  113300                                     8          613    13700       638  24200                                      9          617    45200       668  55500                                      10         613    39600       652  53300                                      11         504    10200       539  12600                                      12         542    39600       596  42000                                      13         612    25000       657  32600                                      14         577    42400       623  39300                                      15         594    34400       638  34000                                      16         592    34200       638  27400                                      17         572    53100       606  74600                                      18         635    31600       675  84600                                      19         613    22670       650  18160                                      20                            639                                             21         588    76290       633  94150                                      22         607     9300       648  42350                                      23         638    61500       658  66900                                      24         629    17950       660  28850                                      26         631    34620       683  35220                                      27         552    28940       585  39450                                      28         564    24210       604  41760                                      29         613    24870       648  37780                                      30         593    13990       631  22850                                      31.1       590    40970       623  51170                                      31.2       587    36950       619  46620                                      32         656    48450       709  134420                                     ______________________________________                                         .sup.1) TBHB = 2,4,6tribromo-3-hydroxybenzoic acid                            .sup.2) aniline = Nmethyl-N-phenylaminomethanephosphonic acid                 hydrochloride                                                                 .sup.3) ext = extinction in [liter/mole cm.sup.2                         

EXAMPLE 34 Determination of creatinine

a) Indicator system 2,4,6-tribromo-3-hydroxybenzoic acid/compound fromExample 4.

Reagent I:

100 mM tris buffer, pH 7.9

200 mM potassium chloride

0.25% detergent (Triton X 100®)

5 mM sodium cholate

10 mM ammonium chloride

5 mM magnesium chloride

15 mM 2,4,6-tribromo-3-hydroxybenzoic acid

15 U/ml. creatinine iminohydrolase (E.C. 3.5.4.21)

0.5 U/ml. N-methylhydantoinase (published Federal Republic of GermanyPatent Specification No. A-3406770)

3 U/ml. sarcosine oxidase (E.C. 1.5.3.1)

3 U/ml. peroxidase (E.C. 1,11.1.7)

Reagent II:

20 mM potassium phosphate buffer, pH 6.0

100 mM ATP

3 mM compound from Example 4

40 U/ml. N-carbamoylsarcosine hydrolase (published Federal Republic ofGermany Patent Specification No. A-3248145)

Sample material:

Aqueous creatinine standard solutions (2 to 20 mg./dl. creatinine).

    ______________________________________                                        Test batch and carrying out:                                                  ______________________________________                                        wavelength           569 mn                                                   layer thickness      10 mm.                                                   temperature          25° C.                                            incubation time      10 minutes                                               ______________________________________                                    

    ______________________________________                                        Pipetting scheme:                                                             ______________________________________                                               Reagent I     1.00 ml.                                                        Reagent II    50 μl.                                                       sample        20 μl.                                                ______________________________________                                    

Measurement against reagent blank (water instead of standard solution assample).

The results obtained are set out in the following Table:

    ______________________________________                                        standard                                                                      concentration  absorption                                                     (mg./dl.)      (mE)                                                           ______________________________________                                         2              92                                                             4             194                                                             6             272                                                             8             368                                                            10             472                                                            12             565                                                            14             658                                                            16             753                                                            18             841                                                            20             974                                                            ______________________________________                                    

b) Indicator system N-methyl-N-phenylaminomethanephosphonic acid(MPA)/compound from Example 4.

Reagent I:

Composition as in the case of Reagent I in a) but with 2 mM MPA(prepared according to published European Patent Specification No.A-0,175,250) instead of 15 mM 2,4,6-tribromo-3-hydroxybenzoic acid.

Reagent II:

Composition identical with Reagent II in a)

Sample material:

Aqueous standard solution (2 to 20 mg./dl. creatinine)

Test batch and carrying out:

Layer thickness, temperature, incubation time, pipetting scheme andmeasurement as in a). Wavelength: 620 nm.

The results obtained are set out in the following Table:

    ______________________________________                                        standard                                                                      concentration  absorption                                                     (mg./dl.)      (mE)                                                           ______________________________________                                         2              61                                                             4             125                                                             6             184                                                             8             249                                                            10             307                                                            12             369                                                            14             429                                                            16             491                                                            18             554                                                            20             615                                                            ______________________________________                                    

c) Indicator system N-ethyl-N-3-sulpho-2-hydroxypropyl-m-anisidine(ADOS)/compound from Example 4.

Reagent I:

Composition as in the case of Reagent I in a) but with 2 mM ADOS (seeChem. Pharm. Bull., 30, 2492/1982) instead of 15 mM2,4,6-tribromo-3-hydroxybenzoic acid.

Reagent II:

Composition identical with Reagent II in a).

Sample material:

Aqueous standard solutions (2 to 20 mg./dl. creatinine).

Test batch and carrying out:

Layer thickness, temperature, incubation time, pipetting scheme andmeasurement as in a)

Wavelength: 598 nm.

The results obtained are set out in the following Table:

    ______________________________________                                        standard                                                                      concentration  absorption                                                     (mg./dl.)      (mE)                                                           ______________________________________                                         2              48                                                             4             107                                                             6             165                                                             8             227                                                            10             280                                                            12             338                                                            14             386                                                            16             450                                                            18             500                                                            20             565                                                            ______________________________________                                    

EXAMPLE 35. Determination of glucose in serum or whole blood by means ofa test carrier.

The reagents necessary for the detection are incorporated into a filmcoating mass according to published European Patent Specification No.A-0,016,387:

0.2 mole/liter phosphate buffer, pH 5.5

0.2 mMole/liter sompound of Example 3

1.0 mMole/liter anilinemethane phosphonic acid (according publishedEuropean Patent Specification No. A-0,175,250)

1.0 KU/liter glucose oxidase

10.0 KU/liter peroxidase.

The film mass is raked out with a layer thickness of 150 μm. on paper asa porous carrier. For the film formation, the mass is left to dry forabout minutes at 50° C.

For the determination of the glucose concentration (0 to about 35mMole/liter) of serum or whole blood applied to the film, after about 1minute the remission is measured on one side by means of a photometer.As the following Table of values shows, a precise determination of theglucose concentration can readily be carried out via a previouslyproduced calibration curve.

    ______________________________________                                        Table of values                                                               glucose (mg./dl.)                                                                             remission (%)                                                 ______________________________________                                         0              93                                                             11             91                                                            103             71                                                            205             51                                                            302             39                                                            400             31                                                            506             25                                                            598             21                                                            ______________________________________                                    

EXAMPLE 36 Determination of hydrogen peroxide in an aqueous solution

Reagent solutions:

1.75 mMole/liter compound from Example 19

2.0 mMole/liter anilinemethanephosphonic acid (according to publishedEuropean Patent Specification No. A-0,175,250)

1.25 KU/liter peroxidase (activity determination withtetramethylbenzidine)

100 mMole/liter phosphate buffer, pH 5.5.

For the production of a calibration curve, 2.28 ml. of phosphate bufferare placed in 1 cm. cuvettes and, in each case, 100 μl. of compound fromExample 22 and anilinomethanephosphonic acid, as well as 10 μl.peroxidase solution pipetted therein. The sample solution is obtained bythe addition of 10 μl. of a definite hydrogen peroxide solution. Thereference solution is obtained by the addition of the same volume ofphosphate buffer. In each case, 30 seconds after the addition of thehydrogen peroxide, the extinction is measured of the coloured materialformed by oxidative coupling as function of the amount of peroxide usedat λ_(max) =660 nm at 25° C., using an Uvikon apparatus.

The values summarised in the following Table are in accordance with theLamber-Beer Law over the whole of the measurement range. The correlationcoefficient of the regression lines amounts to 0.99985. The hydrogenperoxide solutions used correspond to glucose concentrations in thediagnostic relevant range of from 30 mg./dl. to 600 mg./dl.

                  TABLE                                                           ______________________________________                                        Extinction values of the coupling product as function of                      the hydrogen peroxide concentration                                           mMole/liter                                                                   hydrogen                                                                      peroxide       extinction                                                     ______________________________________                                        1.74           0.132                                                          3.48           0.232                                                          6.96           0.448                                                          10.44          0.622                                                          13.92          0.858                                                          17.40          1.051                                                          20.88          1.253                                                          24.36          1.463                                                          27.84          1.635                                                          31.32          1.842                                                          34.80          2.061                                                          ______________________________________                                    

It will be understood that the specification and examples areillustrative but not limitative of the present invention and that otherembodiments within the spirit and scope of the invention will suggestthemselves to those skilled in the art.

We claim:
 1. A method for colorimetric determination of hydrogen peroxide or hydrogen peroxide-forming systems, peroxidase or peroxidate-active substances by means of oxidative coupling of an electron-rich aromatic compound with a heterocyclic compound wherein the electron-rich aromatic compound enters into an oxidative coupling with p-phenylene diamine comprising a 3-amino pyrazolo heterocyclic derivative for color formation selected from the formulas consisting of II, III, IV, V, VI, VII, VIII, IX, X, XI, XII and XIII as follows: ##STR47## wherein --X--Y is --N═CR² and whereinR² is alkyl, alkenyl, alkoxy, alkylthio, aryl or aralkyl or wherein R² is unsubstituted or substituted by hydroxyl, dialkylphosphinyl, carboxyl, SO₃ H, PO₃ H₂, a salt of one of the acid residues or by amino, which amino is unsubstituted or substituted by one or two alkyls wherein said one or two alkyls are unsubstituted or substituted by hydroxyl, carboxyl or alkoxycarbonyl, and wherein when the amino is substituted by two alkyls these two alkyls are unjoined or are joined to form a ring which, apart from the first nitrogen atom of the amino group, are uninterrupted or interrupted by oxygen, sulphur or a second nitrogen atom, or the amino is unsubstituted or substituted by one or two from the group consisting of acyl, alkoxy, aralkoxycarbonyl, H₂ N--CO--, alkyl, aralkyl and arylcarbamoyl; or R² is hydrogen, carboxyl, alkoxycarbonyl, carboxamido or halogen; and wherein R³ is alkyl or aralkyl, and R⁴, R⁵, R⁶ and R⁷ each are hydrogen, alkyl, alkoxy, alkylthio, hydroxyl, aralkyl, aryl, carboxyl, carboxyamido, alkoxycarbonyl, cyano or amino, which amino is unsubstituted or substituted by one or two alkyls wherein said one or two alkyls are unsubstituted or substituted by one or two from the group consisting of hydroxyl, carboxyl, alkoxycarbonyl and halogen; or two substituents on neighboring carbon atoms of a carbon chain form an alkylene radical which alkylene radical is unsubstituted or substituted or anellated with aryl, and tautomers and salts thereof.
 2. The method of claim 1 wherein the 3-aminopyrozolo derivative is selected from the group of compounds consisting of formulas (II), (III), (IV), (VI), (VII), (IX), and tautomers and salts thereof.
 3. The method of claim 1 wherein the electron-rich aromatic compound is a phenol or an aniline compound.
 4. The method of claim 1 wherein the pyrazolo compound is 3-amino-2 methylpyrazolo [1,5-a]pyridine and salts thereof.
 5. An reagent for colorimetric determination of hydrogen peroxide, hydrogen peroxide-forming systems, peroxidase or peroxidate-active substances by oxidative coupling, comprising an electron-rich aromatic compound and a heterocyclic compound, wherein the heterocyclic compound is a 3-aminopyrazolo derivative of claim
 1. 6. The reagent of claim 5 wherein the electron-rich aromatic compound is a phenol or an aniline compound.
 7. The reagent of claim 5 wherein a compound selected from the group of compounds which enter into an oxidative coupling with p-phenylenediamine is used as the electron-rich aromatic compound.
 8. The reagent of claim 5 wherein the pyrazolo compound is 3-amino-2 methylpyrazolo [1,5-a]pyridine and salts thereof.
 9. A method for the preparation of a reagent for the colorimetric determination of hydrogen peroxide, hydrogen peroxide-forming systems, peroxidase or peroxidate-active substances by oxidative coupling wherein the reagent comprises the 3-aminopyrazolo heterocyclic derivative of claim
 1. 10. A process for the colorimetric determination of an election-rich aromatic compound by oxidative coupling thereof with a heterocyclic compound in the presence of an oxidation reagent wherein the heterocyclic compound is the 3-aminopyrazolo heterocyclic derivative of claim
 1. 11. A reagent for the colorimetric determination of an electron-rich aromatic compound by oxidative coupling comprising a heterocyclic compound and an oxidation agent wherein the heterocyclic compound is a pyrazolo derivative of claim
 1. 12. A method for the preparation of a reagent for colorimetric determination of an electron-rich aromatic compound by oxidative coupling wherein the reagent comprises a 3-aminopyrazolo heterocyclic derivative of claim
 1. 